Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.     

体细胞重编程为多能干细胞的研究进展

胚胎干细胞不仅是研究哺乳动物早期胚胎发育、细胞分化、基因表达调控等发育生物学问题的有力工具,还可用于新药评价、细胞治疗等方面的研究.然而,为科学研究而捐献的人类卵子并不能够轻易获得,限制了人类胚胎干细胞相关研究的进展,解决这个问题的理想办法就是找到能够替代胚胎干细胞的其他成体多能细胞.综述了将哺乳动物体细胞诱导为多能干细胞的方法,重点介绍了利用特定的转录因子将体细胞诱导为诱导多能干细胞(induced pluripotent stem cells,iPS细胞)的最新进展,详细阐述了转录因子在诱导细胞重编程过程中发挥的作用,以及iPS细胞筛选与鉴定的方法,并展望了iPS细胞的应用前景.     

脂肪来源间充质干细胞体外定向诱导分化血管细胞的研究进展

脂肪来源的间充质干细胞具有较强的体外增殖能力,因具有生物学特性稳定、来源充足、体外培养条件低等优势已引起各国学者的关注。脂肪间充质干细胞凭借其多向分化潜能,是人体干细胞库潜在的重要来源之一。目前,研究人员已成功地在体外将其诱导为内皮细胞,成骨细胞、成软骨细胞、脂肪前体细胞、平滑肌细胞,心肌细胞、神经样细胞等。就脂肪来源的间充质干细胞体外定向诱导分化血管细胞的研究进展作一综述。     

MIP-3α 联合成骨诱导因子诱导大鼠脂肪干细胞向成牙本质样细胞分化*

目的:观察巨噬细胞炎性蛋白-3α(MIP-3α)对大鼠脂肪干细胞(Adipose derived stem cells,ASCs)向成牙本质样细胞体外分化作用的影响。方法:分离、培养并鉴定大鼠ASCs;以MIP-3α联合成骨诱导因子(地塞米松,β-甘油磷酸钠,以及抗坏血酸)诱导第3代大鼠ASCs向成牙本质样细胞定向分化。诱导培养1、4、7d后,分别测定碱性磷酸酶(alkaline phosphatese,ALP)活性,并用RT-PCR及Western Blot检测成牙本质细胞的标志基因dspp及标志物牙本质涎蛋白(DSP)。结果:与单独加入成骨诱导因子相比,MIP-3α与成骨诱导因子联合应用能使ALP活性、dspp的mRNA表达以及DSP升高。结论:本研究显示MIP-3α与成骨诱导因子联合应用可以增强成牙本质细胞相关基因以及蛋白的表达,为牙齿再生种子细胞的寻找开辟了一条新思路。     

体细胞重编程为诱导多能干细胞

诱导多功能性干细胞（induced pluripotent stem cells,iPS细胞）是通过导入特定的转录因子（如Oct3/4、Sox2、c-Myc和Klf4等）将体细胞诱导重编程为多能性干细胞,其功能与胚胎干细胞相似.iPS细胞的建立,在生命科学领域引起了新的轰动.目前,iPS细胞的研究领域在转录因子的优化、iPS细胞的筛选、载体的运用、体细胞种类的选择和iPS细胞的应用等方面取得突破进展,但仍然存在致癌性、效率低等一系列急需解决的问题.     

Differentiation of Human Embryonic Stem Cells into Cells with Corneal Keratocyte Phenotype

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.     

体细胞重编程为诱导多能干细胞的研究

通过逆转录病毒将4个基因(Oct4 、 Sox2、c-Myc和Klf4)导入小鼠胚胎成纤维细胞 (mouse embryonic fibroblast, MEF)中,能诱导形成胚胎干细胞样特性的诱导多能干(induced pluripotent stem, iPS)细胞.人类iPS细胞的成功构建开拓了广泛的应用前景.本文简要综述了 iPS细胞的基因筛选,转导基因的选择以及iPS细胞的表观遗传特性等.     

Direct Reprogramming of Rat Neural Precursor Cells and Fibroblasts into Pluripotent Stem Cells

Background

Given the usefulness of rats as an experimental system, an efficient method for generating rat induced pluripotent stem (iPS) cells would provide researchers with a powerful tool for studying human physiology and disease. Here, we report direct reprogramming of rat neural precursor (NP) cells and rat embryonic fibroblasts (REF) into iPS cells by retroviral transduction using either three (Oct3/4, Sox2, and Klf4), four (Oct3/4, Sox2, Klf4, and c-Myc), or five (Oct3/4, Sox2, Klf4, c-Myc, and Nanog) genes.

Methodology and Principal Findings

iPS cells were generated from both NP and REF using only three (Oct3/4, Sox2, and Klf4) genes without c-Myc. Two factors were found to be critical for efficient derivation and maintenance of rat iPS cells: the use of rat instead of mouse feeders, and the use of small molecules specifically inhibiting mitogen-activated protein kinase and glycogen synthase kinase 3 pathways. In contrast, introduction of embryonic stem cell (ESC) extracts induced partial reprogramming, but failed to generate iPS cells. However, when combined with retroviral transduction, this method generated iPS cells with significantly higher efficiency. Morphology, gene expression, and epigenetic status confirmed that these rat iPS cells exhibited ESC-like properties, including the ability to differentiate into all three germ layers both in vitro and in teratomas. In particular, we found that these rat iPS cells could differentiate to midbrain-like dopamine neurons with a high efficiency.

Conclusions/Significance

Given the usefulness of rats as an experimental system, our optimized method would be useful for generating rat iPS cells from diverse tissues and provide researchers with a powerful tool for studying human physiology and disease.     

microRNA在多能干细胞向心肌细胞分化中的作用

microRNAs(miRNAs)是长约22 nt的非编码RNAs,广泛参与细胞的增殖、分化、病变、修复和凋亡等多种生命活动．多能干细胞（pluripotent stem cells）是指体外具有自我更新和多向分化潜能的细胞,在一定条件下可被定向诱导分化为多种细胞类型．miRNAs在多能干细胞中表达丰富,并通过调控基因表达影响其自我更新及分化．由多能干细胞向心肌细胞分化的方法主要有3种,即拟胚体形成法、与内胚层细胞共培养法和特定诱导物添加法．虽然这3种方法均可成功诱导多能干细胞向心肌细胞分化,但重复率很低. 所以,人们把研究的视野逐渐转向miRNAs--这个广泛参与细胞生命活动的小分子物质．大量研究表明,在多能干细胞中,不同的miRNAs可通过打靶不同基因影响其向心肌细胞分化．在间充质干细胞中,miR-1、miR-133 和miR-499可分别打靶Hes-1、SRF和Pdcd4| 而在胚胎干细胞中,miR-1和miR-499分别打靶 Hand2和Pacs2促进其向心肌细胞分化．miRNAs在多能干细胞向心肌分化作用机制的研究必将促进再生医学在心脏疾病治疗上的应用．     

c-Myc对诱导性多能干细胞的诱导效率及全能性影响的研究

该研究探讨转录因子c—Myc对多能诱导干细胞（inducedpluripotentstemcells,iPS）诱导效率及形成的iPS克隆全能性的影响。将Yam,dnaka四因子（Oct4、Sox2、Klf4和C—Myc,OSKM）和不舍C。Myc的三因子（OSK）病毒分别感染OG2小鼠成纤维细胞（mouseembryofibroblast,MEF）,诱导成为iPSN胞,通过计数iPS克隆形成数目和流式细胞仪分析iPS克隆的绿色荧光蛋白（GFP）阳性比例。比较OSKM和OSK诱导iPS的效率。分别挑取三株OSK和OSKM诱导的iPS克隆,采用荧光定量PCR法、碱性磷酸酶fAP）染色法和免疫荧光法检测iPS克隆的干性相关蛋白的表达;采用畸胎瘤实验、嵌合体形成实验和四倍体补偿实验检测iPS克隆的全能性。结果显示：在该实验室诱导体系里,OSK病毒感染MEF后形成的iPS克隆数目明显增多,GFP阳性克隆的比例增加。OSK和OSKM诱导iPS克隆的干细胞基本特征,包括形态、AP染色、干细胞特异性基因表达、三胚层形成均没有明显差异。然而,OSK诱导iPS克隆在形成嵌合体小鼠的全能性评估实验中的阳性比例高于OSKM克隆,且只有OSK克隆株能够获得生殖系传递小鼠和四倍体补偿小鼠。该研究表明,在实验诱导体系里去除C—Myc的三因子OSK诱导iPS的效率显著高于OSKM四因子,且三因子诱导iPS克隆具有更好的全能性。     

基于人多潜能干细胞的人类疾病研究与治疗

人多潜能干细胞(hPSC)包括人胚胎干细胞(hESC)和诱导性多潜能干细胞(hiPSC),理论上具有分化成为人类所有细胞类型的能力.基于hPSC的基因打靶技术,不但可以纠正人基因组中的遗传突变用于细胞治疗,还可以通过反向遗传学的方式向hPSC引入疾病特异的突变.将携带人类疾病遗传基因的hPSC分化为特定的细胞类型,在理论上可以在体外模拟人类疾病的发生,研究人类疾病发生的机理,并建立体外筛选平台寻找治疗性药物.基因编辑和干细胞技术的结合将为人类疾病的机制研究和再生医学治疗带来革命性的突破.     

Occludin-deficient Embryonic Stem Cells Can Differentiate into Polarized Epithelial Cells Bearing Tight Junctions

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.     

诱导胚胎干细胞心肌定向分化的方法及其机制

胚胎干细胞是具有分化为各种类型组织细胞潜能的全能干细胞,可在体外大量扩增,细胞因子、激素、诱导剂和细胞内转录因子等可诱导和调控胚胎干细胞进行心肌细胞定向分化．这将使干细胞移植治疗心肌损伤性疾病成为可能。该文介绍胚胎干细胞定向心肌分化的诱导因素及其机制的研究进展。     

诱导多潜能干细胞（iPSCs）：安全高效的诱导策略

在分化的体细胞中,导入特定的转录因子能诱导得到诱导多潜能干细胞(induced pluripotent stem cells,iPSCs).iPSCs在细胞形态、生长特性、表面标志物以及畸胎瘤形成等方面与胚胎干细胞(embryonic stem cells,ESCs)非常相似,而且跟ESCs相比, iPSCs具有避免免疫排斥和不涉及伦理问题的优势,因此iPSCs的临床应用潜力巨大.然而,iPSCs具有成瘤性,而且其诱导效率极低,此二者严重阻碍了iPSCs的临床应用.为解决这两大难题,本综述将主要探讨安全高效的iPS细胞诱导策略,以期为促进其临床应用提供借鉴.     

Repression of SIRT1 Promotes the Differentiation of Mouse Induced Pluripotent Stem Cells into Neural Stem Cells

The use of transplanting functional neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) has increased for the treatment of brain diseases. As such, it is important to understand the molecular mechanisms that promote NSCs differentiation of iPSCs for future NSC-based therapies. Sirtuin 1 (SIRT1), a NAD⁺-dependent protein deacetylase, has attracted significant attention over the past decade due to its prominent role in processes including organ development, longevity, and cancer. However, it remains unclear whether SIRT1 plays a role in the differentiation of mouse iPSCs toward NSCs. In this study, we produced NSCs from mouse iPSCs using serum-free medium supplemented with retinoic acid. We then assessed changes in the expression of SIRT1 and microRNA-34a, which regulates SIRT1 expression. Moreover, we used a SIRT1 inhibitor to investigate the role of SIRT1 in NSCs differentiation of iPSCs. Data revealed that the expression of SIRT1 decreased, whereas miRNAs-34a increased, during this process. In addition, the inhibition of SIRT1 enhanced the generation of NSCs and mature neurocytes. This suggests that SIRT1 negatively regulated the differentiation of mouse iPSCs into NSCs, and that this process may be regulated by miRNA-34a.